Project Summary/Abstract One of the most important knowledge gaps in HIV/SIV vaccine research relates to the molecular properties of viruses that are responsible for mucosal transmission and the initial virus-host cell interactions that lead to productive viral infection. The present proposal addresses this priority area by taking advantage of our laboratory's recent discovery of a novel strategy for identifying mucosally transmitted HIV-1 and SIV viral genomes (Keele, PNAS 2008; Keele, J Exp Med 2009). This strategy, based on single genome amplification, sequencing and analysis of plasma viral RNA within the context of a model of random virus evolution, identifies those viruses that are actually responsible for transmission and productive infection. This innovation, in turn, makes possible for the first time the molecular cloning and biological analysis of actual transmitted/founder SIV viruses. In this application, we propose to extend this new approach to the SIV-macaque infection model and to test the following hypothesis: Molecular clones of full-length SIVsmE660 and SIVmac251 viral genomes corresponding to transmitted/founder viruses can be identified, will be shown to be infectious and replication competent, and will recapitulate pathogenic infection in Indian rhesus macaques. Such clones represent novel molecular reagents with which to decipher the earliest virus-host cell interactions responsible for mucosal SIV transmission and can provide new molecularly-defined virus challenge strains for transmission, pathogenesis and vaccine research. Specific aims of the project are organized into R21 (Aims #1-3) and R33 (Aims #4-5) phases amenable to a go/no-go decision. Aims are: 1) To identify transmitted/founder SIVsmE660 and SIVmac251 viruses responsible for establishing productive clinical infection following low-dose mucosal (ir and ivag) inoculation; 2) To molecularly clone full-length transmitted/founder SIVsmE660 and SIVmac251 proviral genomes; 3) To biologically characterize molecular clone-derived SIVsmE660 and SIVmac251 virus strains compared with SIVsmE660 and SIVmac251 virus isolates in vitro with respect to replication efficiency, cell tropism, and envelope phenotype; 4) To determine infectivity, replication kinetics, and pathogenicity of cloned SIVsmE660 and SIVmac251 virus strains in Indian rhesus macaques following low-dose mucosal inoculation; 5) To characterize molecular pathways of virus diversification and adaptation between transmission and the establishment of set-point viremia as a prelude to homologous and heterologous vaccine-challenge studies. Results from these studies promise to shed new light on the molecular basis of mucosal transmission by SIVsmE660 and SIVmac251, identify new potential targets for protective vaccine-elicited immune responses, and provide much needed molecular clones of genetically-diverse, mucosally-transmitted SIV strains for transmission, pathogenesis and vaccine research.